IL-2, a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan et al., 1976, Science 193:1007-1008. Then called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes, it is now recognized that in addition to its growth factor properties it modulates a variety of functions of immune system cells in vitro and in vivo and has been renamed IL-2. IL-2 is one of several lymphocyte-produced messenger-regulatory molecules that mediate immunocyte interactions and functions.
IL-2 was initially made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2-producing cell lines. See, for instance, U.S. Pat. No. 4,401,756. Recombinant DNA technology has provided an alternative to PBLs and cell lines for producing IL-2. Taniguchi et al., 1983, Nature 302:305-310; U.S. Pat. No. 4,738,927, issued Apr. 19, 1988; and Devos, 1983, Nucleic Acids Research11:4307-4323 have reported cloning the human IL-2 gene and expressing it in microorganisms.
Native human IL-2 is an antigen-nonspecific, genetically unrestricted soluble factor produced by erythrocyte rosette positive T cells stimulated with antigens, mitogens or alloantigens. It is a protein with a reported molecular weight in the approximate range of 13,000 to 17,000 daltons (S. Gillis and J. Watson, 1980, J Exp Med 159:1709) and an isoelectric point in the approximate range of pH 6-8.5.
Human IL-2 has a number of in vitro and in vivo effects including enhancing the proliferative responses of human peripheral blood mononuclear cells or murine thymocytes, enhancing the immune response in humans and in animals against bacterial, parasitic, fungal, protozoan and viral infections, and supporting the growth of continuous T cell lines.
Human IL-2 has been obtained from genetically engineered E. coli as an unglycosylated protein with biological activities equivalent to those of native, glycosylated IL-2. (Taniguchi et al., 1983, Nature 11:4307-4323; Rosenberg et al., 1984, Science 223:1412-1415; Wang et al., 1984, Science224:1431-1433; and Doyle et al., 1985, J Biol Resp Modifiers 4:96-109). Rosenberg and his coworkers have shown that systemic administration of recombinant IL-2 in high doses causes regression of established metastatic cancers in mice (Rosenberg et al., 1985, J Exp Med 161:1169-1188); and, in conjunction with lymphokine-activated killer cells (Rosenberg et al., 1985, New Eng J Med 313:1485-1492) and tumor-infiltrating lymphocytes (Rosenberg et al., 1986, Science 233:1318-1321), in humans.
U.S. Pat. No. 4,518,584 discloses muteins (analogs) of IL-2 in which the cysteine normally occurring at position 125 of the wild-type or native molecule has been replaced with a neutral amino acid, such as serine or alanine. European Patent (EP) publication 200,280 discloses muteins of IL-2 whereby the methionine at position 104 has been replaced by a conservative amino acid.
Microbially produced IL-2 is not glycosylated and is primarily produced in a denatured state. It is largely insoluble and, when expressed at high levels, it precipitates intracellularly in the form of "refractile" or "inclusion" bodies which appear as bright spots visible within the cell under a phase contrast microscope at magnifications down to 1000-fold. The problem addressed by the present invention is how to efficiently recover the IL-2 from the cell in a purified, cystine-bridged, renatured form that is acceptable for clinical use.
The heretofore available methods for recovering microbially produced IL-2 are described below.
U.S. Pat. No. 4,569,790 describes a process for recovering recombinant IL-2 from an IL-2-producing microorganism in which the cell is disrupted, the disruptate is extracted with an aqueous solution of a chaotropic agent such as urea, the IL-2 is solubilized with a surfactant, e.g., sodium dodecyl sulfate (SDS), and the IL-2 is separated in the presence of a reducing agent.
Commonly owned U.S. Pat. Nos. 4,530,787 and 4,572,978 described processes for purifying recombinant IL-2 from microorganisms in which partially purified reduced IL-2 is selectively oxidized under controlled conditions to its oxidized (cystine) form. The former patent uses o-iodosobenzoic acid as an oxidizing agent and the latter uses Cu+2 cation as an oxidation promoter.
European Patent publication 206,828 published 30 Dec. 1986, and entitled "Process for Recovering Refractile Bodies Containing Heterologous Proteins from Microbial Hosts" discloses methods for recovering and purifying refractile bodies of IL-2 from E. coli. To isolate the refractile material, the processes initially involve disrupting the cell wall and membrane of the host cell, removing greater than 99% by weight of the salts from the disruptate, redisrupting the desalted disruptate, adding a material to the disruptate to create a density or viscosity gradient in the liquid within the disruptate, and separating the refractile material from the cellular debris by high-speed centrifugation. The IL-2 is then solubilized with a solubilizing agent such as SDS, chromatographed to remove high molecular weight contaminants, oxidized, and purified by a combination of HPLC, ultrafiltration and gel filtration.
An abstract titled "Purification and Renaturation of Recombinant Interleukin-2" presented at the 6th International Symposium on HPLC of Proteins, Peptides and Polynucleotides at Baden-Baden, West Germany in October 1986 describes a process in which recombinant IL-2 is solubilized from inclusion bodies with 6M guanidine hydrochloride/10 mM dithiothreitol (DTT) and purified in a reduced, denatured form by FPLC gel permeation. The solution from the FPLC gel permeation is diluted to effect renaturation and autoxidation. In this regard U.S. Pat. Nos. 4,511,502, 4,511,503, 4,512,922, and 4,518,526, and EP publication 114,506 describes a similar procedure for purifying heterologous proteins in general from refractile bodies. In such processes, the oxidation and renaturation of the IL-2 are carried out in a single step. However, because of disparate solubility characteristics between the reduced and oxidized forms of IL-2, it is difficult to achieve high yields of renatured oxidized IL-2 in such a process.
EP publication 145,390 describes a process for recovering rIL-2 from E. coli in which the cells are suspended in 7M guanidine hydrochloride, solids are removed by centrifugation, the rIL-2-containing supernatant is dialyzed to remove the guanidine hydrochloride and the dialyzate is purified by anion exchange chromatography, gel filtration and RP-HPLC.
The present invention is directed to an improved recombinant IL-2 purification process in which the oxidation and renaturation are carried out in distinct steps.